When recombinantly expressing peptides such as antibodies, vaccines or other therapeutic polypeptides (e.g., interferon or erythropoietin), plant-derived hydrolysates are often added to the cell culture media in order to increase the titer. The small peptide chains in the hydrolysates are derived from protease-driven degradation of soy and wheat by-products of the food industry which results in the unintended introduction of proteases to the culture. The presence of the proteases leads to in culture degradation of the antibodies, particularly if engineered to be secreted into the culture. Later purification is also complicated if the proteases are not sufficiently inactivated or removed and are, instead, carried over, in an active form, to the purified product. Moreover, due to the irregular nature of the plant sources from which the hydrolysates are derived, lot-to-lot variability may also be observed. The variability makes removal or inactivation of the proteases more difficult.
During manufacture of the hydrolysates, an enzyme, often a papain-derivative, is added to a batch of soy, wheat, or rice. The enzyme is allowed to digest the food material for a specific amount of time and is then, typically, heat inactivated via a pasteurization step. For high-throughput production, heat inactivation of the enzyme is achieved using heat exchangers. The contact time, however, is minimal. For this reason, complete inactivation is not always successful. Typically, the hydrolysates are then ultrafiltered with membranes ranging in size cut-off from 10 kDa to 50 kDa. Some enzymes are not removed during this step due to inefficiency associated with ultrafiltration when used for removal of polypeptides from a sample. Leaching of the digestion enzyme into the product has been documented in the literature (Mols et al., Biotechnology Letters 26: 1043-1046 (2004)) wherein a thiol-protease was discovered in rice hydrolysates from CWBI (Centre Wallon de Biologie Industrielle) that had been ultrafiltered.
The presence of even very low levels of proteases in a peptide product can cause significant problems with respect to the quality and stability of the product over time. Slow proteolytic degradation of the product is a problem that must be monitored in order to both track product quality and stability and to develop purification procedures for complete removal of protease contamination from the product. There are known methods for detecting proteases in a sample, however, the sensitivity of such assays is relatively low. For example, culture media vendors commonly use a fluorescence-based assay with a casein peptide substrate that emits fluorescence light when cleaved by a protease. Since the quantity of protease present in a sample of purified antibody product is, generally, very low, the known protease assays are insufficient. There exists a need in the art for protease assays comprising sensitivity high enough to detect very low levels of protease activity in a sample.